IGEM:MIT/2006/Notebook/2006-6-20: Difference between revisions

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==To do==
==To do==
#Set up and run gradient PCR for BAMT and SAMT
#Possibly reconsider BAMT/SAMT primers
#run test gels on exhaustive BAMT and SAMT pcr products - possibly reconsider primers
#Reconsider ATF1 primers definitely
#Reconsider ATF1 primers definitely
#Grow colonies on sketchy minimal media plates
#Smell LB liquid and minimal liquid cultures
#Pour new minimal plates
#Get in touch with GC lab
#Get in touch with GC lab
#****3 pm: Stephen has meeting --- light sensor part!
#****3 pm: Stephen has meeting --- light sensor part!
==WINTERGREEN!!!!!!! (and plating)==
#The liquid cultures from yesterday '''SMELLED LIKE WINTERGREEN!!'''  SO EXCITING!!!! YAY!
#The SAMT+SA and the BSMT+SA worked, in both LB and minimal media
#*Now it's time to optimize SA concentration :)
#Plated liquid cultures onto M9 with 50, 100, 200 μL SA
#*2 sets of 4 (BSMT M9, BSMT LB, SAMT M9, SAMT LB) with one set of 3 missing BSMT M9
==Indole==
#Indole from [[../2006-6-19 |6-19-2006]] definitely was root of bad bacterial smell.
==Repeat Smell Experiment==
Each tube has 20 uL cells + 10 mL LB Kan + Salicylic Acid unless otherwise noted
'''Controls'''
BL21 + LB (no Kan) + 6.2 uL SA + Induced
SAMT + 31.0 uL SA
SAMT + 6.2 uL SA
SAMT + 3.1 uL SA
BSMT + 31.0 uL SA
BSMT + 6.2 uL SA
BSMT + 3.1 uL SA
'''Experimental'''
SAMT + 31.0 uL SA + Induced
3 Tubes X (SAMT + 6.2 uL SA + Induced
SAMT + 3.1 uL SA + Induced
BSMT + 31.0 uL SA + Induced
3 Tubes X (BSMT + 6.2 uL BSMT + Induced)
BSMT + 3.1 uL SA + Induced
==Repeat BAMT/SAMT PCR and run PCR products on gels==
#16 sample tubes, 6 controls (8, 3 of each)
#*(-): no template- just primers, (-): no primer- just plasmid, (+): new primer/template
#Sample master mix 18x, control master mix 10x, [[../2006-6-15 | from here]]
#Temperature gradient from 42-54
#PCR was a failure, must repeat at higher temperature
==Repeat ATF1 PCR at higher temperature range==
#try 48 to 64 degrees
#place most of tubes in center of PCR machine
==BSMT PCR cleanup and digest==
#cut with ECOR1 and PST1

Latest revision as of 13:41, 20 June 2006

To do

  1. Possibly reconsider BAMT/SAMT primers
  2. Reconsider ATF1 primers definitely
  3. Get in touch with GC lab
          • 3 pm: Stephen has meeting --- light sensor part!

WINTERGREEN!!!!!!! (and plating)

  1. The liquid cultures from yesterday SMELLED LIKE WINTERGREEN!! SO EXCITING!!!! YAY!
  2. The SAMT+SA and the BSMT+SA worked, in both LB and minimal media
    • Now it's time to optimize SA concentration :)
  3. Plated liquid cultures onto M9 with 50, 100, 200 μL SA
    • 2 sets of 4 (BSMT M9, BSMT LB, SAMT M9, SAMT LB) with one set of 3 missing BSMT M9

Indole

  1. Indole from [[../2006-6-19 |6-19-2006]] definitely was root of bad bacterial smell.

Repeat Smell Experiment

Each tube has 20 uL cells + 10 mL LB Kan + Salicylic Acid unless otherwise noted

Controls

BL21 + LB (no Kan) + 6.2 uL SA + Induced

SAMT + 31.0 uL SA

SAMT + 6.2 uL SA

SAMT + 3.1 uL SA

BSMT + 31.0 uL SA

BSMT + 6.2 uL SA

BSMT + 3.1 uL SA

Experimental

SAMT + 31.0 uL SA + Induced

3 Tubes X (SAMT + 6.2 uL SA + Induced

SAMT + 3.1 uL SA + Induced

BSMT + 31.0 uL SA + Induced

3 Tubes X (BSMT + 6.2 uL BSMT + Induced)

BSMT + 3.1 uL SA + Induced

Repeat BAMT/SAMT PCR and run PCR products on gels

  1. 16 sample tubes, 6 controls (8, 3 of each)
    • (-): no template- just primers, (-): no primer- just plasmid, (+): new primer/template
  2. Sample master mix 18x, control master mix 10x, [[../2006-6-15 | from here]]
  3. Temperature gradient from 42-54
  4. PCR was a failure, must repeat at higher temperature

Repeat ATF1 PCR at higher temperature range

  1. try 48 to 64 degrees
  2. place most of tubes in center of PCR machine

BSMT PCR cleanup and digest

  1. cut with ECOR1 and PST1
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